1. Field of the Invention
This invention relates to a method for cloning and copying genetic material on surfaces as well as copying biological material insofar as it can be classified in a broader sense in a ligand-receptor system.
2. Background Information
Methods of exponentially amplifying molecular matrices are already known through the work of G. von Kiedrowski et al. (Nature 1998, Vol. 346, 245-248; Federal Republic of Germany Patent No. 198 48 403). The amplification cycles are characterized by:
binding of molecular matrices to the surface of a solid phase by means of a reversible linker on the matrix;
addition of matrix fragments, with one of the fragments displays a linker unit, which may, if necessary, be protected;
synthesizing copies of the matrix;
removal of superfluous matrix fragments and ancillary reaction substances;
detachment of the copies from the matrix; and
application of synthesized matrix copies to free binding sites on the solid phase.
This represents an iterative, progressive amplification method, allowing an exponential increase in the amount of molecular matrices available, thus enabling a significant process of evolution to take place. To achieve this the process makes use of the surface of a solid carrier. Chemical attachment to immobilized matrices enables copies to be synthesized from precursor matrices, which are then released to become new matrices. This process can be repeated any number of times.
In addition, so-called xe2x80x9cbridgexe2x80x9d amplification technology is described in U.S. Pat. No. 5,641,658. This is an amplification model based on conventional PCR methods, but which is intended to achieve localized amplification. Bridge amplification technology has many uses, especially in analytical methods that can also be carried out with the commonly used PCR. The bridge technology facilitates the separation and detection stages of the amplified products. The characteristics of this technology are that it combines amplification, selection and detection in a single process. Advanced state of the art systems can be found on the homepage of MOSAIC Technologies, Inc. (USA), the company marketing bridge amplification technology (www.mostek.com).
The bridge system describes a method for amplifying nucleic acids on a solid phase, with both amplification primers being bound covalently to a single solid phase through their 5xe2x80x2 ends. Consequently this represents a further development of the well-known polymerase chain reaction, known as PCR for short. This takes place in a solid phase PCR instead of in a solution. The particular advantage of this method is its ability to amplify and analyze many different genetic elements simultaneously using a single sample. The applications for bridge amplification technology include genetic expression, genome research, clinical diagnostics and the examination of biological fluids, e.g. blood. A higher rate of amplification is achieved by eliminating ineffective primer artifacts (such as primer dimers). This enables simple, sensitive and cost-effective DNA detection methods to be developed, for example using fluorescence. Because bridge amplification technology ensures that all amplification products remain bound to the solid phase, contamination through prolongation remains low, and this in turn enhances the diagnostic value of the method compared with the usual PCR.
Whereas the method proposed by G. von Kiedrowski et al. referred to above demonstrates the benefits of the solid phase amplification of entire populations, the method described in U.S. Pat. No. 5,641,658 offers the advantage of amplifying a single matrix on a solid phase. However, the drawback of this bridge amplification method is linked with the problem of product inhibition, i.e. a newly produced copy may occur not only with the adjacent immobilized primer, but also with the original matrix strand, which is also adjacent. Another disadvantage is the lower linear limitation needed to achieve bridging as a double strand. Furthermore, there is no separation between the strands, with the result that, for diagnostic purposes, the hybridization signals are weakened due to hybridization with complementary strands.
Federal Republic of Germany Patent No. 694 09 646 T2 describes a method for amplifying a nucleic acid, in which the one primer is bound to a solid phase and the second primer to a particle that reacts with a magnetic field. These primers are incorporated in target nucleic acid sequences. Following an extension stage the nucleic acid strands are separated by the application of an electric current. The magnetic primer can be particle-bonded, existing as a form of solid phase. The avidin/biotin system is suitable for binding the primer to the solid phases. This method is also suitable for cloning.
U.S. Pat. No. 5,795,714 describes a method which, in one form, uses an array of oligonucleotides, which are connected to the surface of the solid phases by means of the reciprocal reaction between biotin and avidin. The method described consists of the hybridization of complementary strands, primer extension reactions, the hybridization of a second biotinylized primer to the primer extension products, and the extension of the second primer. Mention is made of the blotting of copies on a second surface which is coated with avidin.
The disadvantage of the methods referred to above is that multiple replication in the sense of exponential propagation is not possible, and that the translocation of the copies by means of an electromagnetic field cannot be achieved without loss of site information. Based on this state of the art, and avoiding the shortcomings referred to above, it is therefore the task of this invention to provide a method of cloning and copying onto surfaces which permits the propagation of biological material while retaining site information.
The present invention overcomes the problems and disadvantages of current amplifications methods and enables biological systems including, nucleic acids, ligands and receptors, to be propagated and separated from one another by means of an electric field for immobilization and fixation on one or more solid surfaces, while retaining site information.
The present invention considers a biological system to be basically an interaction between nucleic acids of any kind and/or with peptides/proteins/polymerases/enzymes (DNA/RNA/PNA/pRNA/2xe2x80x2-5xe2x80x2 nucleotides and RNA/DNA mirror mers (see PCT/EP97/04726)), in exactly the same way as antigen/antibody complexes or, in general terms, ligand/receptor systems.
For a basic understanding of the invention it is necessary to appreciate that complementary nucleic acids themselves represent nothing more than a special form of a complementary ligand/receptor system in a traditional sense. For purposes of the present application, the term xe2x80x9cligandxe2x80x9d will refer to one molecule of the biological binding pair and the term xe2x80x9creceptorxe2x80x9d will refer to the opposite molecule of the biological binding pair. Two complementary strands of nucleic acid are biological binding pairs. One of the strands is designated the ligand and the other strand is designated the receptor. Biological binding pairs may also comprise antigen and antibodies, drugs and drug receptor sites and enzymes and enzyme substrates.
The invention utilizes the fact that, because of their charge, nucleic acids and many other biologically relevant molecules can be moved within an electrical field when such a field is applied. In the present case a stationary, bound molecule is separated from a corresponding molecule in this way, in that the non-stationary, bound molecule is either separated from the other by synthesization of that molecule or following an xe2x80x9cidentification reactionxe2x80x9d, with the aid of an electrical field. Because the molecules tend to migrate along the line of the electrical field, they retain site information while migrating. This is also what mainly distinguishes the method referred to in this invention from the previously known state of the art, because in Federal Republic of Germany Patent No. 694 09 646 T2, for example, site information is not retained, since the electromagnetic field described in that case does not serve to retain the site information.
For all of the alternative processes shown hereinbelow it is possible to alter, reduce and/or increase the (geometrical) scale of the transfer, while retaining site information, within the respective transfer stage of the respective process sequence. This may be practical, for example, if the geometry of the arrays from which or to which the transfer is being made is not identical with that of the initial array or target array.
The present invention in one aspect relates broadly to a method for propagating ligands and receptors on at least two surfaces, comprising:
(a) immobilizing a first ligand on a first surface of a substantially solid phase;
(b) adding a solution of receptors and binding complementary receptors to the first ligand;
(c) transferring the receptor to a second surface and immobilizing the receptor at that location;
(d) attaching an additional ligand to the immobilized receptor; and
(e) transferring the additional ligand to the first surface and immobilizing it at that location, wherein the steps set forth above may be repeated multiple times.
Preferably, the surface in step (c) is a second surface which is spatially separated from the first. The additional ligand is transferred to the second surface by the application of an electrical field, and preferably the electrical field is applied between the first and second surface.
Another aspect of the invention relates to enzymatic propagation of a nucleic acid sequence on at least two surfaces, comprising:
(a) immobilizing a first primer on at least one first surface of a substantially solid phase;
(b) administering a solution of nucleic acids comprising complementary fragments to the first primer;
(c) binding of complementary fragments to the first primer;
(d) extending the first primer at its 3xe2x80x2 end, corresponding to the complementary fragment by means of a polymerase;
(e) releasing the complementary fragments;
(f) attaching a second primer to the 3xe2x80x2 end of the extended nucleic acid;
(g) extending the second primer at its 3xe2x80x2 end by means of a polymerase;
(h) transferring the second primer to another surface and immobilization of the extended primer; and
(i) attaching another first primer to the 3xe2x80x2 end of the second extended primer for further extending of the first primer, wherein the steps of (b) to (h) are repeated numerous times for exponential amplification of nucleic acid sequence.
The second primer as set forth in step (h) is transferred to a second surface which is spatially separated from the first, and the transfer is achieved by the application of an electrical field. Preferably, the electrical field is applied between the first and second surface.
Further amplification stages may occur following the transferal of the second primer to the second surface comprising the following:
(j) extending this first primer to its 3xe2x80x2 end, corresponding to the complementary fragment, by means of a polymerase;
(k) transferring of the extended primer to the first or another surface and immobilization of the extended primer thereon; and
(l) attaching of another second primer to the 3xe2x80x2 end of the extended first primer.
A further aspect of the invention relates to a method for copying nucleic acids from a first to a second surface, comprising:
(a) immobilizing of nucleic acids through a reaction on a carrier surface;
(b) producing a double-stranded molecule by a method selected from the group consisting of hybridization of complementary single strands, chemical or enzymatic ligation of complementary fragments and chemical or enzymatic extension of complementary primers; and
(c) transferring of complementary strands to a second surface with immobilization of the complementary strands thereon.
The transfer of the complementary strands to the second surface may be accomplished by the application of an electrical field, in which the electrical field is applied between the first and second surface.
Preferably, the nucleic acids immobilized on the solid carrier are arranged two-dimensionally and are transferred in this order, while retaining site information. The solid phase material is selected from organic or inorganic material or from a hybrid of these materials, and preferably represents a two- or three-dimensional matrix. Immobilization of the nucleic acids and complementary strands occurs through covalent or non-covalent binding.
In still another aspect, the nucleic acids, ligands, receptors or their derivatives are provided with a detectable label. Generally, any molecular moiety capable of detection may be utilized including, by way of example, without limitation radioisotopes, stable isotopes, enzymes, immunoreactive compounds, fluorescence or luminescence chemicals, chromophores, metals or charged particles.
The present invention may be applicable for several different amplification methods including, cloning genomic fragments of DNA, cDNA and RNA, subcloning following restriction-digesting, strengthening an immunological ligand/receptor pair, strengthening the ligand signal, sorting adjacent fragments by using hybridization techniques (chromosome walking), and copying of gene chips.
The term xe2x80x9camplificationxe2x80x9d is used in the broad sense to mean creating a product which may include, by way of example, additional target molecules, or target-like molecules or molecules complementary to the target molecules, which molecules are created by virtue of the presence of the target molecule in the sample. In a situation where the target is a nucleic acid, an amplification product can be made enzymatically with an agent for polymerization, such as with DNA or RNA polymerases or transcriptases.
The above-discussed embodiments of the present invention will be described further hereinbelow. When the word xe2x80x9cinventionxe2x80x9d is used in this specification, the word xe2x80x9cinventionxe2x80x9d includes xe2x80x9cinventionsxe2x80x9d, that is the plural of xe2x80x9cinventionxe2x80x9d. By stating xe2x80x9cinventionxe2x80x9d, the Applicant does not in any way admit that the present application does not include more than one patentably and non-obviously distinct invention, and maintains that this application may include more than one patentably and non-obviously distinct invention. The Applicant hereby asserts that the disclosure of this application may include more than one invention, and, in the event that there is more than one invention, that these inventions may be patentable and non-obvious one with respect to the other.